Drug induced toxicity remains one of the major reasons for the failure of drug candidates to be approved and the withdrawal of approved drugs from the market. See, e.g., Olson et al., Regul. Toxicol. Pharmacol. (2000) 32:56-67. Chemically reactive electrophilic metabolites of the drug are likely mediators of the toxicity, possibly by acting as covalent modifiers of essential cellular machinery. See, e.g., Guengerich et al., Arch. Biochem. Biophys. (2005) 433:369-378; Kalgutkar et al., Curr. Drug. Metab. (2005) 6:161-225. Often drugs undergo biotransformation to metabolites that can interfere with cellular functions through their intrinsic chemical reactivity towards glutathione (GSH), leading to GSH depletion, and towards other functionally critical macromolecules, resulting in reversible modification, irreversible adduct formation, or irreversible loss of activity. See, e.g., Srivastava et al., Handb. Exp. Pharmacol. (2010) 196:165-194. There is now a great deal of evidence which shows that reactive metabolites are formed from drugs known to cause hepatotoxicity, such as acetaminophen, tamoxifen, isoniazid, and amodiaquine.
Preclinical screens have been developed in an effort to minimize bioactivation liabilities in the early stages of drug discovery. See, e.g., Ma and Subramanian, J. Mass. Spectrom. (2006) 41:1121-1139. The most common analytical techniques used in pre-clinical screens are gas chromatography (GC) or liquid chromatography (LC) coupled to mass spectrometry (MS), e.g., such as GC or LC coupled to tandem mass spectrometry (MS/MS) scanning. Mass spectrometry offers a much greater sensitivity than alternative methods, such as nuclear magnetic resonance (NMR) spectroscopy, and thus affords the analysis of numerous low abundance metabolites, but its quantitative precision is inherently poorer. One strategy for improving the detection of metabolites by mass spectrometry involves treating the sample with a “heavy” and “light” version of an isotopic labeling reagent, thereby creating a “heavy” and “light” version of the labeled metabolite. See, e.g., Lamos et al., Anal. Chem. (2007) 79:5143-5149. Installing a positively-charged functional group has also been found to enhance the ion efficiency and corresponding high detection sensitivity in positive ion mode electrospray ionization-mass spectrometry (ESI-MS). See, e.g., Lamos in supra, Yang et al., Anal. Chem. (2006) 78:4702-4708; Johnson, Rapid Commun. Mass. Spectrom. (2000) 14:2019-2024; Barry et al., Rapid Commun. Mass. Spectrom. (2003) 17:603-620; Mirzaei et al., Anal. Chem. (2006) 78:4175-4183; Soglia et al., Chem. Res. Toxicol. (2006) 19:480-490; and U.S. Patent Application No. 2004/0248234.
However, despite these efforts, there continues to remain a need for additional improvement and development of early screening assays to identify and/or quantify potential chemically reactive electrophilic metabolites which may be responsible for drug-induced toxicity.